Isolation of non-embryonic stem cells and uses thereof

Patent metadata

Field	Value
Publication number	US-2016237400-A1
Application number	US-201514865494-A
Country	US
Kind code	A1
Filing date	Sep 25, 2015
Priority date	Mar 15, 2013
Publication date	Aug 18, 2016
Grant date	—

Abstract

The invention described herein relates to methods of isolating non-embryonic stem cell, e.g., adult stem cell, from a non-embryonic tissue, e.g., an adult tissue or organ. Non-embryonic stem cells (e.g., adult stem cells) thus isolated from the various tissues or organs can self-renew or propagate indefinitely in vitro, are multipotent and can differentiate into the various differentiated cell types normally found within the tissue or organ from which the stem cells are isolated. In addition, the isolated stem cells can be propagated through clonal expansion of a single isolated stem cell, to produce a clone of which at least about 40%, 70%, or 90% or more cells within the clone can be further passaged as single cell originated clones.

First claim

1 . A method for isolating a non-embryonic stem cell from a non-embryonic tissue, the method comprising:
(1) culturing dissociated epithelial cells from the non-embryonic tissue, in contact with a first population of lethally irradiated feeder cells and a basement membrane matrix, to form epithelial cell clones, in a medium comprising:
(a) a Notch agonist;
(b) a ROCK (Rho Kinase) inhibitor;
(c) a Bone Morphogenetic Protein (BMP) antagonist;
(d) a Wnt agonist;
(e) a mitogenic growth factor; and,
(f) insulin or IGF (or an agonist thereof);
said medium optionally further comprising at least one of:
(g) a TGFβ receptor inhibitor; and,
(h) nicotinamide or an analog thereof;
(2) isolating single cells from said epithelial cell clones, and,
(3) culturing isolated single cells from step (2) individually to form single cell clones, in contact with a second population of lethally irradiated feeder cells and a second basement membrane matrix in said medium;
wherein each of said single cell clones represents a clonal expansion of said non-embryonic stem cell, thereby isolating said non-embryonic stem cell.

2 - 4 . (canceled)

5 . The method of claim 1 , further comprising isolating single non-embryonic stem cell from said single cell clones, and optionally further comprising culturing one of said single cell clones to generate a pedigree cell line of said non-embryonic stem cell.

6 - 9 . (canceled)

10 . The method of claim 1 , wherein the non-embryonic tissue is obtained from or originates in lung, esophagus, stomach, small intestine, colon, intestinal metaplasia, fallopian tube, kidney, pancreas, bladder, or liver, or a portion/section thereof.

11 . The method of claim 1 , wherein the non-embryonic tissue is a disease tissue, a disorder tissue, an abnormal condition tissue, or a tissue from a patient having said disease, disorder, or abnormal condition, such as, for example, wherein the disease, disorder, or abnormal condition comprises an adenoma, a carcinoma, an adenocarcinoma, a cancer, a solid tumor, an inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), ulcer, gastropathy, gastritis, oesophagitis, cystitis, glomerulonephritis, polycystic kidney disease, hepatitis, pancreatitis, an inflammatory disorder (e.g., type I diabetes, diabetic nephropathy) and autoimmune disorder.

12 - 14 . (canceled)

15 . The method of claim 1 , wherein in step (1) the (epithelial) cells are dissociated from the non-embryonic tissue through enzymatic digestion with an enzyme, such as collagenase, protease, dispase, pronase, elastase, hyaluronidase, accutase and/or trypsin.

16 . (canceled)

19 . The method of claim 1 , wherein the basement membrane matrix is a laminin-containing basement membrane matrix (e.g., MATRIGEL™ basement membrane matrix (BD Biosciences)), preferably growth factor-reduced, preferably wherein the basement membrane matrix does not support 3-dimensional growth, or does not form a 3-dimensional matrix necessary to support 3-dimensional growth.

20 - 21 . (canceled)

22 . The method of claim 1 , characterized by one or more of the following:
the Notch agonist comprises Jagged-1, the ROCK inhibitor comprises Rho Kinase Inhibitor VI (Y-27632, (R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide)), Fasudil or HA1071 (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), or H1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride), the BMP antagonist comprises Noggin, DAN, a DAN-like proteins comprising a DAN cystine-knot domain (e.g., Cerberus and Gremlin), Chordin, a chordin-like protein comprising a chordin domain, Follistatin, a follistatin-related protein comprising a follistatin domain, sclerostin/SOST, decorin, or α-2 macroglobulin, the Wnt agonist comprises R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, an R-spondin mimic, a Wnt family protein (e.g., Wnt-3a, Wnt 5, Wnt-6a), Norrin, or a GSK-inhibitor (e.g., CHIR99021), the mitogenic growth factor comprises EGF (and/or Keratinocyte Growth Factor, TGFα, BDNF, HGF, bFGF), the TGFβ receptor inhibitor comprises SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide), A83-01, SB-505124, SB-525334, LY 364947, SD-208, or SJN 2511, and/or, the TGFβ (signaling) inhibitor binds to and reduces the activity of one or more serine/threonine protein kinases selected from the group consisting of ALK5, ALK4, TGF-beta receptor kinase 1 and ALK7.

23 - 29 . (canceled)

30 . The method of claim 1 , wherein the medium comprises: 5 μg/mL insulin; 2 nM of (3,3′,5-Triiodo-L-Thyronine); 400 ng/mL hydrocortisone; 24.3 μg/mL adenine; 10 ng/mL EGF; 10% fetal bovine serum (without heat inactivation); 1 μM Jagged-1; 100 ng/mL noggin; 125 ng/mL R-spondin 1; 2.5 μM Y-27632; and 1.35 mM L-glutamine in DMEM:F12 3:1 medium, optionally further comprising 0.1 nM cholera enterotoxin, and optionally further comprises one or more of 2 μM SB431542 and 10 mM nicotinamide.

31 - 37 . (canceled)

38 . The method of claim 30 , wherein the non-embryonic tissue is fetal small intestine, and the medium further comprises: FGF receptor inhibitor; N-Acetyl-L-cysteine; a p38 inhibitor (e.g., SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-796); Gastrin; PGE2; an FGF receptor inhibitor; Shh; TGFβ; 10 mM nicotinamide and TGFβ; 10 mM nicotinamide and Wnt3a; 10 mM nicotinamide and GSK3 inhibitor; or 10 mM nicotinamide and 2 μM SB431542.

39 . The method of claim 1 , wherein the medium lacks at least one of: Wnt3a, p38 inhibitor (e.g., SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-796), N-Acetyl-L-cysteine, Gastrin, HGF, testosterone (e.g., (dihydro)testosterone), N2, B27, and PGE2.

40 . (canceled)

41 . The method of claim 1 , wherein said non-embryonic stem cell, when isolated as single cell, is capable of self-renewal for greater than about 50 generations, 70 generations, 100 generations, 150 generations, 200 generations, 250 generations, 300 generations, 350 generations, or about 400 or more generations.

42 . (canceled)

43 . The method of claim 1 , wherein said non-embryonic stem cell is a small intestine stem cell, and is capable of differentiating into a differentiated small intestine cell that (1) expresses a marker selected from MUC or PAS (goblet cell markers), CHGA (neuroendocrine cell marker), LYZ (Paneth cell marker), MUC7, MUC13, and KRT20; and/or (2) absorbs water and nutrients (such as by differentiated enterocytes), secretes mucus (such as by differentiated goblet cells), secretes intestinal hormones (such as by differentiated enteroendocrine cells), or secreting antibacterial substances (such as by differentiated Paneth cells).

44 . The method of claim 1 , wherein said non-embryonic stem cell expresses one or more stem cell markers selected from: SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A and preferably said non-embryonic stem cell is a small intestine stem cell, and expresses one or more markers selected from: OLFM4, SOX9, LGR5, CLDN18, CA9, BPIFB1, KRT19, CDH17, and TSPAN8.

45 . (canceled)

46 . The method of claim 1 , wherein said non-embryonic stem cell substantially lacks expression of marker(s) associated with differentiated cell types in said non-embryonic tissue, preferably said non-embryonic stem cell is a small intestine stem cell, and lacks expression of markers associated with differentiated s

Assignees

• Jackson Lab

Inventors

• Xian Wa

Classifications

• A61K35/38 Primary

  Stomach; Intestine; Goblet cells; Oral mucosa; Saliva · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/11

  Epidermal growth factor [EGF] · CPC title

• C12N2500/38

  Vitamins · CPC title

• A61K35/30

  Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue · CPC title